Evaluating the conduct and reporting of the T-cell dependent antibody response in the Extended One-Generation Reproductive Toxicity study provided under the EU REACH regulation.

The European Union (EU) Chemicals Strategy for Sustainability regards chemicals that affect the immune system among the most harmful ones. The Extended One-Generation Reproductive Toxicity study (EOGRTS; Organisation for Economic Co-Operation and Development (OECD) Test Guideline (TG) 443), addresses, among others, potential effects of chemicals on development. In specific cases, the EOGRTS is performed with addition of a so-called cohort 3, that addresses potential effects on the developing immune system, by means of a central assay measuring the T-cell dependent antibody response (TDAR). This assay is based on an interplay of antigen presentation, T-cell help and antibody production by B-cells, and together comprises a functional immune response. In the context of the EOGRTS review project of the European Chemicals Agency (ECHA), we evaluated 15 available TDARs for compliance with conduct and reporting requirements. Collectively, the majority of the TDAR studies were considered to be adequately conducted. We however observed: (i) the protocols differed by the antigen used (sheep red blood cells (SRBC) or KLH), the route of administration (intravenous, intraperitoneal, or subcutaneous), prime or prime/boost immunizations, and whether IgG was measured. (ii) There was major variation in the effects of the positive control for immunosuppression, cyclophosphamide. (iii) Proficiency was not always shown. (iv) Statistical analysis was not always done or reported. (v) Results of effects on lymphocyte populations or other immunotoxicity observations obtained in cohort 1 (or 2) of the EOGRTS were not always discussed together with results of the TDAR. Taken together, next to an improved quality of reporting, this may suggest a need to better define the conduct of the TDAR in OECD TG 443 and OECD Guidance Document (GD) 151, at least for certain aspects.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS). In Tier 0, before entering the Thyroid-NDT-TAS, all available in vivo, in vitro and in silico data are submitted to weight-of-evidence (WoE) evaluations to determine whether the substance of interest poses a concern for thyroid disruption. If so, Tier 1 of the Thyroid-NDT-TAS includes an initial MoA and human relevance assessment (structured by the key events of possibly relevant adverse outcome pathways) and the generation of supportive in vitro/in silico data, if relevant. Only if Tier 1 is inconclusive, Tier 2 involves higher-tier testing to generate further thyroid- and/or neurodevelopment-related data. Tier 3 includes the final MoA and human relevance assessment and an overarching WoE evaluation to draw a conclusion on whether, or not, the substance meets the EDC-T. The Thyroid-NDT-TAS is based on the state-of-the-science, and it has been developed to minimise animal testing. To make human safety assessments more accurate, it is recommended to apply the Thyroid-NDT-TAS during future regulatory assessments.

Investigations on learning and memory function in extended one-generation reproductive toxicity studies - when considered needed and based on what?

To justify investigations on learning and memory (L&M) function in extended one-generation reproductive toxicity studies (EOGRTS; Organization for Economic Co-operation and Development (OECD) test guideline (TG) 443) for registration under Registration, Evaluation, Authorization, and Restriction of Chemical (REACH), the European Chemicals Agency has referred to three publications based on which the Agency concluded that "perturbation of thyroid hormone signaling in offspring affects spatial cognitive abilities (learning and memory)" and "Therefore, it is necessary to conduct spatial learning and memory tests for F1 animals". In this paper, the inclusion of the requested L&M tests in an EOGRTS is challenged. In addition, next to the question on the validity of rodent models in general for testing thyroid hormone-dependent perturbations in brain development, the reliability of the publications specifically relied upon by the agency is questioned as these contain numerous fundamental errors in study methodology, design, and data reporting, provide contradicting results, lack crucial information to validate the results and exclude confounding factors, and finally show no causal relationship. Therefore, in our opinion, these publications cannot be used to substantiate, support, or conclude that decreases in blood thyroid (T4) hormone level on their own would result in impaired L&M in rats and are thus not adequate to use as fundament to ask for L&M testing as part of an EOGRTS.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny-part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects. Based upon the rat data reviewed, maternal serum thyroid hormone levels do not show a causal relationship with statistically significant neurodevelopmental effects. Offspring serum thyroxine together with offspring serum triiodothyronine and thyroid stimulating hormone appear relevant to predict the likelihood for neurodevelopmental effects. Based upon the collated database, thresholds of ≥60%/≥50% offspring serum thyroxine reduction and ≥20% and statistically significant offspring serum triiodothyronine reduction indicate an increased likelihood for statistically significant neurodevelopmental effects; accuracies: 83% and 67% when excluding electrophysiology (and gene expression). Measurements of brain thyroid hormone levels are likely relevant, too. The extent of substance-mediated thyroid hormone imbalance appears more important than substance mode-of-action to predict neurodevelopmental impairment in rats. Pertinent research needs were identified, e.g. to determine whether the phenomenological offspring thyroid hormone thresholds are relevant for regulatory toxicity testing. The insight from this review shall be used to suggest a tiered testing strategy to determine whether gestational/lactational substance exposure may elicit thyroid hormone imbalance and potentially also neurodevelopmental effects.

Unnecessary use of additional animals for determination of sexual maturation in the EOGRTS.

The Extended-One-Generation Study [EOGRTS, OECD 443] is a study in which the toxic effects of test substances on reproduction (Cohort 1), neurodevelopment (Cohort 2), and development of the immune system (Cohort 3) in rats are evaluated. The latter two Cohorts are not always required according to the European Chemicals Agency (ECHA) based on data from previously performed toxicity studies. Although the Cohorts for developmental neurotoxicity (DNT) and developmental immunotoxicity (DIT) are often omitted, the F1-animals normally required for these Cohorts are still maintained for evaluation of sexual maturation since three F1-animals/sex/litter/group are required according to OECD Guidance Document (GD) No. 151. This review investigates whether two F1-animals/sex/litter/group would suffice for this endpoint by investigating the rationale provided by the GD and by comparing results of eighteen EOGRTSs in which three versus two F1-animals/sex/litter/group were evaluated. After a comprehensive literature research, we concluded that the rationale in the GD does not substantiate the decision to use three F1-animals/sex/litter/group. The scientific papers provided as rationale focused on male observations and the observations discussed do not match the observations for sexual maturation mentioned by the guidelines. The investigation using data from eighteen EOGRTSs showed that the toxicological conclusions, whether the test substance affected sexual maturation or not, matched when comparing data of two F1-animals/sex/litter/group to three F1-animals/sex/litter/group. To conclude, two F1-animals/sex/litter/group would suffice for the evaluation of sexual maturation, which negates the requirement for a so called "Cohort 1 C" (i.e. 160 animals (80 males and 80 females)) per EOGRTS, as well as the number of regulated procedures that need to be performed.

Adverse postnatal developmental effects in offspring from humidifier disinfectant biocide inhaled pregnant rats.

Inhalation exposure to polyhexamethylene guanidine phosphate (PHMG-P), one of the primary biocides used in humidifier disinfectants, caused a fatal pulmonary disease in Korea. Pregnant women were also exposed to PHMG-P, and subsequent studies showed that PHMG-P inhalation during pregnancy adversely affects their health and embryo-fetal development. However, the postnatal developmental effects after birth on prenatally PHMG-P-exposed offspring have not yet been investigated. Therefore, in this study, we aimed to examine the postnatal development of prenatally PHMG-P-exposed offspring. Pregnant rats (22 or 24 females per group) were exposed to PHMG-P during pregnancy in a whole-body inhalation chamber at the target concentrations of 0, 0.14, 1.60, and 3.20 mg/m3. After parturition, the prenatally exposed offspring were transferred to non-exposed surrogate mothers to minimize the secondary effects of severe maternal toxicities. Postnatal development of offspring was then examined with a modified extended one-generation reproductive toxicity study design. At 3.20 mg/m3 PHMG-P, increased perinatal death rates and decreased viability index (postnatal survival of offspring between birth and postnatal day 4) were observed. In addition, F1 offspring had lower body weight at birth that persisted throughout the study. PHMG-P-exposed pregnant rats also had severe systemic toxicities and increased gestation period. At 1.60 mg/m3 PHMG-P, a decreased viability index was also observed with systemic toxicities of PHMG-P-exposed pregnant rats. These results indicate that prenatal PHMG-P exposure adversely affects the offspring's future health and could be used for human risk assessment.

Extended One-Generation Reproductive Toxicity (EOGRT) study of benzoic acid in Sprague Dawley rats.

Benzoic acid (BA) was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats in an OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study to test for effects that may occur as a result of pre- and postnatal exposure. The study included cohorts of F1 offspring to evaluate potential effects of benzoic acid on reproduction, the developing immune system, and the developing neurological system with the inclusion of learning and memory assessments. Benzoic acid was incorporated in the diet at concentrations of 0, 7,500, 11,500, and 15,000 mg/kg diet (ppm). These concentrations were selected based on the results of preliminary studies, and, based on average food consumption, were intended to supply BA doses of approximately 0, 500, 750, and 1000 mg/kg bw/day. To avoid exceeding these target dose levels, the dietary concentrations were adjusted (based on historical control body weight and food consumption data) to maintain the target mg/kg bw/day dose levels during those life periods when food intake per unit of body weight was increased to support milk production by females (gestation and lactation) and rapid pup growth (post-weaning). In the parental (F0) generation, survival, clinical observations, organ weights, pathology, hematology, serum chemistry, urinalysis, and bile acids were unaffected by BA administration. Reproductive parameters were also unaffected by BA administration. In the F1 generation, survival, growth and developmental landmarks, organ weights, pathology, immunotoxicity assessment, and neurotoxicity and neurobehavioral parameters such as auditory startle response, locomotor activity, learning and memory assessments were unaffected by BA administration, as were clinical pathology (hematology, serum chemistry, urinalysis, bile acids and thyroid hormones) and reproductive performance. Similarly, no adverse effects or systemic toxicity were observed in the F2 generation. Overall, the highest dietary concentration (15,000 ppm), providing a dosage of approximately 1000 mg/kg bw/day, was the NOAEL for benzoic acid in this EOGRT study.

Effects of endocrine disruptor furan on reproductive physiology of Sprague Dawley rats: An F1 Extended One-Generation Reproductive Toxicity Study (EOGRTS).

The present study investigated the reproductive toxicity of furan in an Extended One-Generation Reproductive Toxicity Study in rats. Sprague Dawley F0 weaning rats (30 per sex per group) were exposed to furan orally at 0, 1, 2.5, 5, and 10 mg kg-1 for 10 weeks (males) and 2 weeks (females) and then mated. Results of F0 indicated that in the furan-treated groups (5 mg kg-1 and 10 mg kg-1), body weight (bw) gain decreased during prebreed and gestational period while increased during lactation periods. F0 animals prebreeding exposure resulted in head tilt and foot splay at 10 mg kg-1. Number of live pups at birth were decreased (p < 0.001) at 10 mg kg-1. At postnatal day (PND) 70, a significant (p = 0.03) decrease in testosterone levels of male rats and estrogen levels of female rats (p = 0.05) was observed in 10 mg kg-1 furan-treated group in F1 generation. Luteinizing hormone, follicle-stimulating hormone, and progesterone levels were also reduced, but their reduction was not statistically significant in all groups. In higher dose furan group (10 mg kg-1), testicular and ovarian weights were reduced in F1 generation at PND 70, with decreased daily sperm production (p = 0.01) and disturbed estrous cyclicity (p < 0.01). Some histopathological changes were also observed in testis and ovaries in groups whose parents were previously exposed to 10 mg kg-1 bw of furan group. Based on the above results, it is suggested that exposure to food-based contaminant furan induced remarkable changes in the F0 (parental stage) and F1 (offspring, pubertal, and adult stage) generations of Sprague Dawley rats.

Is there a rationale for direct dosing of chemicals to nursing pups in the EOGRTS (OECD 443)?

In OECD guideline 443 - Extended One Generation Reproductive Toxicity Study (EOGRTS) - to be used for testing industrial and agrochemicals, it has been indicated that careful consideration of benefits and disadvantages should be made prior to conducting direct-dosing studies in nursing pups. Nursing pups will not be directly dosed in dietary and drinking water studies whereas in oral gavage studies this possibility exists. Besides the risk of intubation trauma and overdosing due to direct exposure and exposure via the mother's milk, direct dosing could lead to a different hazard assessment of chemicals depending on the choice of the route of administration. In addition, in case of industrial and agrochemicals used in industrial or professional settings only, there will never be direct exposure of newborns. Moreover, direct dosing of nursing pups is an artificial, non-physiological, route of exposure and as such it would hamper risk assessment. It should therefore only be considered in exceptional cases and justified on a case-by-case approach.

Practical considerations for developmental thyroid toxicity assessments: What's working, what's not, and how can we do better?

Thyroid hormones (THs; T3 and T4) play a role in development of cardiovascular, reproductive, immune and nervous systems. Thus, interpretation of TH changes from rodent studies (during pregnancy, in fetuses, neonates, and adults) is critical in hazard characterization and risk assessment. A roundtable session at the 2017 Society of Toxicology (SOT) meeting brought together academic, industry and government scientists to share knowledge and different perspectives on technical and data interpretation issues. Data from a limited group of laboratories were compiled for technical discussions on TH measurements, including good practices for reliable serum TH data. Inter-laboratory historical control data, derived from immunoassays or mass spectrometry methods, revealed: 1) assay sensitivities vary within and across methodologies; 2) TH variability is similar across animal ages; 3) laboratories generally achieve sufficiently sensitive TH quantitation levels, although issues remain for lower levels of serum TH and TSH in fetuses and postnatal day 4 pups; thus, assay sensitivity is critical at these life stages. Best practices require detailed validation of rat serum TH measurements across ages to establish assay sensitivity and precision, and identify potential matrix effects. Finally, issues related to data interpretation for biological understanding and risk assessment were discussed, but their resolution remains elusive.

Opinion on the follow-up of the re-evaluation of sorbic acid (E200) and potassium sorbate (E202) as food additives.

In this opinion, the EFSA Panel on Food Additives and Flavourings (FAF Panel) was requested by the European Commission to carry out a scientific evaluation of an extended one-generation reproductive toxicity study (EOGRTS) to determine whether it would allow reconsideration of the temporary group acceptable daily intake (ADI) for sorbic acid (E 200) and potassium sorbate (E 202), established by the Panel on Food Additives and Nutrient Sources added to Food (ANS Panel) in 2015. From the EOGTRS, the FAF Panel identified a lower confidence limit of the benchmark dose (BMDL) of 1,110 mg sorbic acid/kg body weight (bw) per day. By applying a default uncertainty factor of 100, the Panel established a group ADI expressed as 11 mg sorbic acid/kg bw per day for sorbic acid (E 200) and its potassium salt (E 202). In addition, European Commission asked EFSA to review a report on the 'Stability of sorbic acid (E 200) and its potassium salt (E 202) during food processing and storage' provided by industry. No new information was provided in this report, and therefore, in this opinion, there was no re-assessment of the EFSA ANS opinion conclusions from 2015 regarding the stability of sorbates in food.

Guidance on the selection of cohorts for the extended one-generation reproduction toxicity study (OECD test guideline 443).

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 433) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.

Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations.

In 1998, the OECD initiated a high-priority project aimed at revising existing test guidelines and developing new test guidelines for screening of potential endocrine disruptors. In 2011, OECD 443 was adopted, and in 2015 OECD 421 and OECD 422 were updated with endocrine disruptor relevant endpoints. A feasibility study for the enhancement of OECD 414 with endocrine disruptor relevant endpoints is currently ongoing. The addition of these endpoints is considered crucial for gaining more information on endocrine disruptor potency of tested chemicals, however it should be noted that these additions have a major impact on the study designs and give rise to several practical challenges. The aim of this review is to discuss important aspects of these challenging study designs and to share our knowledge on their implementation in our laboratory. Together, this review can be used as guidance for other laboratories, study monitors and registration officers.

Implementing the extended one-generation reproductive toxicity study (EOGRTS): important points to consider.

The hallmark of the extended one-generation reproductive toxicity study (EOGRTS) is that, based on certain criteria or triggers, selected offspring are assigned at weaning to different cohorts for further investigation of sexual maturation, reproductive organ integrity and function, neuropathological and behavioral endpoints, and/or immune function. The triggers allow for a more customizable design based directly on the data, while minimizing animal usage. Compared to the two-generation reproductive toxicity study, the EOGRTS design increases the number, extent, and duration of F1-offspring assessments resulting in more thorough and efficient utilization of the first generation while excluding the second generation of offspring unless triggered. Therefore, the EOGRTS has the potential to reduce the number of rats required by nearly 1200 animals per study. When performing the EOGRTS, the complexity of this study should not be underestimated and experienced flexible testing laboratories with sufficient resources and historical control data for all parameters are essential. The aim of this review is to discuss the important aspects of this challenging study design and to share our knowledge on the implementation of this study in our laboratories. In addition, we elaborate on the type of criteria for expansion of the study and logistical considerations. Altogether, this review can be used as guidance by other labs, study monitors, and registration officers.

Developmental immunotoxicity of chemicals in rodents and its possible regulatory impact.

Around 25% of the children in developed countries are affected with immune-based diseases. Juvenile onset diseases such as allergic, inflammatory and autoimmune diseases have shown increasing prevalences in the last decades. The role of chemical exposures in these phenomena is unclear. It is thought that the developmental immune system is more susceptible to toxicants than the mature situation. Developmental immunotoxicity (DIT) testing is nowadays not or minimally included in regulatory toxicology requirements. We reviewed whether developmental immune parameters in rodents would provide relatively sensitive endpoints of toxicity, whose inclusion in regulatory toxicity testing might improve hazard identification and risk assessment of chemicals. For each of the nine reviewed toxicants, the developing immune system was found to be at least as sensitive or more sensitive than the general (developmental) toxicity parameters. Functional immune (antigen-challenged) parameters appear more affected than structural (non-challenged) immune parameters. Especially, antibody responses to immune challenges with keyhole limpet hemocyanine or sheep red blood cells and delayed-type hypersensitivity responses appear to provide sensitive parameters of developmental immune toxicity. Comparison with current tolerable daily intakes (TDI) and their underlying overall no observed adverse effect levels showed that for some of the compounds reviewed, the TDI may need reconsideration based on developmental immune parameters. From these data, it can be concluded that the developing immune system is very sensitive to the disruption of toxicants independent of study design. Consideration of including functional DIT parameters in current hazard identification guidelines and wider application of relevant study protocols is warranted.

Regulatory acceptance and use of the Extended One Generation Reproductive Toxicity Study within Europe.

The two-generation study (OECD TG 416) is the standard requirement within REACH to test reproductive toxicity effects of chemicals with production volumes >100 tonnes. This test is criticized in terms of scientific relevance and animal welfare. The Extended One Generation Reproductive Toxicity Study (EOGRTS), incorporated into the OECD test guidelines in 2011 (OECD TG 443) has the potential to replace TG 416, while using only one generation of rats and being more informative. However, its regulatory acceptance proved challenging. This article reconstructs the process of regulatory acceptance and use of the EOGRTS and describes drivers and barriers influencing the process. The findings derive from literature research and expert interviews. A distinction is made between three sub-stages; The stage of Formal Incorporation of the EOGRTS into OECD test guidelines was stimulated by retrospective analyses on the value of the second generation (F2), strong EOGRTS advocates, animal welfare concern and changing US and EU chemicals legislation; the stage of Actual Regulatory Acceptance within REACH was challenged by legal factors and ongoing scientific disputes, while the stage of Use by Industry is influenced by uncertainty of registrants about regulatory acceptance, high costs, the risk of false positives and the manageability of the EOGRTS.

Approaches and considerations for the assessment of immunotoxicity for environmental chemicals: a workshop summary.

As experience is gained with toxicology testing and as new assays and technologies are developed, it is critical for stakeholders to discuss opportunities to advance our overall testing strategies. To facilitate these discussions, a workshop on practices for assessing immunotoxicity for environmental chemicals was held with the goal of sharing perspectives on immunotoxicity testing strategies and experiences, developmental immunotoxicity (DIT), and integrated and alternative approaches to immunotoxicity testing. Experiences across the chemical and pharmaceutical industries suggested that standard toxicity studies, combined with triggered-based testing approaches, represent an effective and efficient approach to evaluate immunotoxic potential. Additionally, discussions on study design, critical windows, and new guideline approaches and experiences identified important factors to consider before initiating DIT evaluations including assay choice and timing and the impact of existing adult data. Participants agreed that integrating endpoints into standard repeat-dose studies should be considered for fulfilling any immunotoxicity testing requirements, while also maximizing information and reducing animal use. Participants also acknowledged that in vitro evaluation of immunosuppression is complex and may require the use of multiple assays that are still being developed. These workshop discussions should contribute to developing an effective but more resource and animal efficient approach for evaluating chemical immunotoxicity.

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10-12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The "No Observed Adverse Effect Level" for systemic toxicity was 300 ppm in both males (16.6 mg/kg/day) and females (20.6 mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.